The general objective is to study the structure of clathrin and associated proteins as a way of understanding the molecular basis of the formation and function of coated pits and coated vesicles in the cell. The proposed experiments involve an integration of structural, biochemical and molecular biological approaches that take advantage of the unusual special features of clathrin. Two parts of the long range project are presented in this proposal. The first part is the determination of the primary structure of calf brain clathrin heavy chain. This work involves cloning the heavy chain cDNA and requires protein chemical and recombinant DNA methodologies. The sequence information will be then used to study the domain organization and its relation to the topological map of the heavy chain. Analysis of domain organization will be carried out at three levels: 1) computer analysis of the primary structure; 2) amino terminal sequence determination of selected proteolytic fragments along the heavy chain; and 3) immunoelectron microscopy mapping of antibodies raised against selected synthetic peptides of the heavy chain. The second part of the project is the analysis of interactions between clathrin and associated proteins that participate in the regulation of coated vesicle formation. We will characterize the oligomeric structure of 100 Kd/50 Kd complex and map its binding site along a clathrin arm. The approach involves chemical crosslinking and single molecule electron microscopy. We will also attempt to establish which subunits of 100 Kd/50 Kd complex are in contact with clathrin.